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The COVID-19 pandemic has prompted an unprecedented global effort to understand and mitigate the spread of the SARS-CoV-2 virus. In this study, we present a comprehensive analysis of COVID-19 in Western New York (WNY), integrating individual patient-level genomic sequencing data with a spatially informed agent-based disease Susceptible-Exposed-Infectious-Recovered (SEIR) computational model. The integration of genomic and spatial data enables a multi-faceted exploration of the factors influencing the transmission patterns of COVID-19, including genetic variations in the viral genomes, population density, and movement dynamics in New York State (NYS). Our genomic analyses provide insights into the genetic heterogeneity of SARS-CoV-2 within a single lineage, at region-specific resolutions, while our population analyses provide models for SARS-CoV-2 lineage transmission. Together, our findings shed light on localized dynamics of the pandemic, revealing potential cross-county transmission networks. This interdisciplinary approach, bridging genomics and spatial modeling, contributes to a more comprehensive understanding of COVID-19 dynamics. The results of this study have implications for future public health strategies, including guiding targeted interventions and resource allocations to control the spread of similar viruses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Bard, Jiang, Emerson, Bartz, Lamb, Marzullo, Pohlman, Boccolucci, Nowak, Yergeau, Crooks and Surtees.)

The kinetoplastid parasite, Trypanosoma brucei , undergoes a complex life cycle entailing slender and stumpy bloodstream forms in mammals and procyclic and metacyclic forms (MFs) in tsetse fly hosts. The numerous gene regulatory events that underlie T. brucei differentiation between hosts, as well as between active and quiescent stages within each host, take place in the near absence of transcriptional control. Rather, differentiation is controlled by RNA-binding proteins (RBPs) that associate with mRNA 3' untranslated regions (3'UTRs) to impact RNA stability and translational efficiency. DRBD18 is a multifunctional T. brucei RBP, shown to impact mRNA stability, translation, export, and processing. Here, we use single-cell RNAseq to characterize transcriptomic changes in cell populations that arise upon DRBD18 depletion, as well as to visualize transcriptome-wide alterations to 3'UTR length. We show that in procyclic insect stages, DRBD18 represses expression of stumpy bloodstream form and MF transcripts. Additionally, DRBD18 regulates the 3'UTR lengths of over 1,500 transcripts, typically promoting the use of distal polyadenylation sites, and thus the inclusion of 3'UTR regulatory elements. Remarkably, comparison of polyadenylation patterns in DRBD18 knockdowns with polyadenylation patterns in stumpy bloodstream forms shows numerous similarities, revealing a role for poly(A) site selection in developmental gene regulation, and indicating that DRBD18 controls this process for a set of transcripts. RNA immunoprecipitation supports a direct role for DRBD18 in poly(A) site selection. This report highlights the importance of alternative polyadenylation in T. brucei developmental control and identifies a critical RBP in this process., Competing Interests: Competing interests statement:The authors declare no competing interest.

The sequencing of human virus genomes from wastewater samples is an efficient method for tracking viral transmission and evolution at the community level. However, this requires the recovery of viral nucleic acids of high quality. We developed a reusable tangential-flow filtration system to concentrate and purify viruses from wastewater for genome sequencing. A pilot study was conducted with 94 wastewater samples from four local sewersheds, from which viral nucleic acids were extracted, and the whole genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was sequenced using the ARTIC V4.0 primers. Our method yielded a high probability (0.9) of recovering complete or near-complete SARS-CoV-2 genomes (>90% coverage at 10× depth) from wastewater when the COVID-19 incidence rate exceeded 33 cases per 100 000 people. The relative abundances of sequenced SARS-CoV-2 variants followed the trends observed from patient-derived samples. We also identified SARS-CoV-2 lineages in wastewater that were underrepresented or not present in the clinical whole-genome sequencing data. The developed tangential-flow filtration system can be easily adopted for the sequencing of other viruses in wastewater, particularly those at low concentrations., Competing Interests: The authors declare no competing financial interest.

Naive human pluripotent stem cells (hPSCs) can be used to generate mature human cells of all three germ layers in mouse-human chimeric embryos. Here, we describe a protocol for generating mouse-human chimeric embryos by injecting naive hPSCs converted from the primed state. Primed hPSCs are treated with a mammalian target of rapamycin inhibitor (Torin1) for 3 h and dissociated to single cells, which are plated on mouse embryonic fibroblasts in 2iLI medium, a condition essentially the same for culturing mouse embryonic stem cells. After 3-4 d, bright, dome-shaped colonies with mouse embryonic stem cell morphology are passaged in 2iLI medium. Established naive hPSCs are injected into mouse blastocysts, which produce E17.5 mouse embryos containing 0.1-4.0% human cells as quantified by next-generation sequencing of 18S ribosomal DNA amplicons. The protocol is suitable for studying the development of hPSCs in mouse embryos and may facilitate the generation of human cells, tissues and organs in animals., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Many surgeons continue to face the clinical dilemma of interpreting a positive aspiration or unexpected positive Cutibacterium acnes (C. acnes) culture. There are factors that complicate the interpretation of positive cultures including variations in both frequency of false positive cultures and virulence properties. As indices of virulence, hemolytic strains, from previously confirmed clinically infected shoulders, were compared with non-hemolytic isolates determined to be contaminants, by RNA-sequencing (RNA-Seq). Six C. acnes isolates from patients who underwent revision total shoulder arthroplasty (TSA) were identified based on previously described infection criteria. Three C. acnes isolates from each group underwent RNA-Seq. Differential gene expression analysis, principal component analysis (PCA), and heatmap analysis were used to determine the gene variation and patterning between the definite infection and probable contaminant isolates. Differential gene expression analysis identified genes that were differentially expressed between the isolates classified as definite infection and isolates classified as probable contaminants. PCA using a 500 gene subset of identified genes was able to find combinations of these genes that separated out the definite infection and probable contaminants isolates. The heatmap demonstrated similar gene expression in the three Definite Infections isolates, and significantly different expression when compared with the probable contaminant isolates. Clinical significance: C. acnes revision TSA isolates classified as definite infection and probable contaminant demonstrated a similar gene expression pattern to each respective group and different gene expression pattern when compared between groups. These findings indicate distinct differences in C. acnes strains associated with clinically relevant orthopedic TSA infections., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

To investigate the genomic evolution of metastatic pediatric osteosarcoma, we performed whole-genome and targeted deep sequencing on 14 osteosarcoma metastases and two primary tumors from four patients (two to eight samples per patient). All four patients harbored ancestral (truncal) somatic variants resulting in TP53 inactivation and cell-cycle aberrations, followed by divergence into relapse-specific lineages exhibiting a cisplatin-induced mutation signature. In three of the four patients, the cisplatin signature accounted for >40% of mutations detected in the metastatic samples. Mutations potentially acquired during cisplatin treatment included NF1 missense mutations of uncertain significance in two patients and a KIT G565R activating mutation in one patient. Three of four patients demonstrated widespread ploidy differences between samples from the sample patient. Single-cell seeding of metastasis was detected in most metastatic samples. Cross-seeding between metastatic sites was observed in one patient, whereas in another patient a minor clone from the primary tumor seeded both metastases analyzed. These results reveal extensive clonal heterogeneity in metastatic osteosarcoma, much of which is likely cisplatin-induced. IMPLICATIONS: The extent and consequences of chemotherapy-induced damage in pediatric cancers is unknown. We found that cisplatin treatment can potentially double the mutational burden in osteosarcoma, which has implications for optimizing therapy for recurrent, chemotherapy-resistant disease., (©2019 American Association for Cancer Research.)

Histone H3 K27M mutation is the defining molecular feature of the devastating pediatric brain tumor, diffuse intrinsic pontine glioma (DIPG). The prevalence of histone H3 K27M mutations indicates a critical role in DIPGs, but the contribution of the mutation to disease pathogenesis remains unclear. We show that knockdown of this mutation in DIPG xenografts restores K27M-dependent loss of H3K27me3 and delays tumor growth. Comparisons of matched DIPG xenografts with and without K27M knockdown allowed identification of mutation-specific effects on the transcriptome and epigenome. The resulting transcriptional changes recapitulate expression signatures from K27M primary DIPG tumors and are strongly enriched for genes associated with nervous system development. Integrated analysis of ChIP-seq and expression data showed that genes upregulated by the mutation are overrepresented in apparently bivalent promoters. Many of these targets are associated with more immature differentiation states. Expression profiles indicate K27M knockdown decreases proliferation and increases differentiation within lineages represented in DIPG. These data suggest that K27M-mediated loss of H3K27me3 directly regulates a subset of genes by releasing poised promoters, and contributes to tumor phenotype and growth by limiting differentiation. The delayed tumor growth associated with knockdown of H3 K27M provides evidence that this highly recurrent mutation is a relevant therapeutic target.

Background: The prevalence and spectrum of predisposing mutations among children and adolescents with cancer are largely unknown. Knowledge of such mutations may improve the understanding of tumorigenesis, direct patient care, and enable genetic counseling of patients and families., Methods: In 1120 patients younger than 20 years of age, we sequenced the whole genomes (in 595 patients), whole exomes (in 456), or both (in 69). We analyzed the DNA sequences of 565 genes, including 60 that have been associated with autosomal dominant cancer-predisposition syndromes, for the presence of germline mutations. The pathogenicity of the mutations was determined by a panel of medical experts with the use of cancer-specific and locus-specific genetic databases, the medical literature, computational predictions, and second hits identified in the tumor genome. The same approach was used to analyze data from 966 persons who did not have known cancer in the 1000 Genomes Project, and a similar approach was used to analyze data from an autism study (from 515 persons with autism and 208 persons without autism)., Results: Mutations that were deemed to be pathogenic or probably pathogenic were identified in 95 patients with cancer (8.5%), as compared with 1.1% of the persons in the 1000 Genomes Project and 0.6% of the participants in the autism study. The most commonly mutated genes in the affected patients were TP53 (in 50 patients), APC (in 6), BRCA2 (in 6), NF1 (in 4), PMS2 (in 4), RB1 (in 3), and RUNX1 (in 3). A total of 18 additional patients had protein-truncating mutations in tumor-suppressor genes. Of the 58 patients with a predisposing mutation and available information on family history, 23 (40%) had a family history of cancer., Conclusions: Germline mutations in cancer-predisposing genes were identified in 8.5% of the children and adolescents with cancer. Family history did not predict the presence of an underlying predisposition syndrome in most patients. (Funded by the American Lebanese Syrian Associated Charities and the National Cancer Institute.).

The class II DNA "cut-and-paste" transposons have been used to efficiently modify the Xenopus genome for transgenesis applications. Once integrated, the transposon is an effective substrate for excision and re-integration (remobilization) elsewhere in the genome by simply supplying the transposase enzyme in trans. We have used two methods to remobilize transposons resident in the frog genome: micro-injection of transposase mRNA at the one-cell stage and expression of the enzyme in the germline from a transgene. Double-transgenic frogs (hoppers) that harbor transgenes for both the substrate transposon and the transposase enzyme are outcrossed to wild-type animals and the progeny are scored for changes in reporter gene expression. Although both methods work effectively to remobilize transposons, the breeding-mediated strategy eliminates the time-consuming micro-injection step; novel integration events are produced by simply outcrossing the hopper frogs. As each outcross of Xenopus tropicalis typically produces 2,000, or more, progeny, this method can be used to perform large-scale insertional mutagenesis screens in this highly tractable developmental model system.

The generation of transgenic animals is an essential tool for many genetic strategies. DNA "cut-and-paste" transposon systems can be used to efficiently modify the Xenopus genome. The DNA transposon substrate, harbored on a circularized plasmid, is co-injected into fertilized Xenopus embryos at the one-cell stage together with mRNA encoding the cognate transposase enzyme. The cellular machinery rapidly translates the exogenous mRNA to produce active transposase enzyme that catalyzes excision of the transposon substrate from the plasmid and stable integration into the genomic DNA.

Background: The Sleeping Beauty (SB) transposon system has been used for germline transgenesis of the diploid frog, Xenopus tropicalis. Injecting one-cell embryos with plasmid DNA harboring an SB transposon substrate together with mRNA encoding the SB transposase enzyme resulted in non-canonical integration of small-order concatemers of the transposon. Here, we demonstrate that SB transposons stably integrated into the frog genome are effective substrates for remobilization., Results: Transgenic frogs that express the SB10 transposase were bred with SB transposon-harboring animals to yield double-transgenic 'hopper' frogs. Remobilization events were observed in the progeny of the hopper frogs and were verified by Southern blot analysis and cloning of the novel integrations sites. Unlike the co-injection method used to generate founder lines, transgenic remobilization resulted in canonical transposition of the SB transposons. The remobilized SB transposons frequently integrated near the site of the donor locus; approximately 80% re-integrated with 3 Mb of the donor locus, a phenomenon known as 'local hopping'., Conclusions: In this study, we demonstrate that SB transposons integrated into the X. tropicalis genome are effective substrates for excision and re-integration, and that the remobilized transposons are transmitted through the germline. This is an important step in the development of large-scale transposon-mediated gene- and enhancer-trap strategies in this highly tractable developmental model system.

Transposon-mediated integration strategies in Xenopus offer simple and robust methods for the generation of germline transgenic animals. Co-injection of fertilized one-cell embryos with plasmid DNA harboring a transposon transgene and synthetic mRNA encoding the cognate transposase enzyme results in mosaic integration of the transposon at early cleavage stages that are frequently passed through the germline in the adult animal. Micro-injection of fertilized embryos is a routine procedure used by many laboratories that use Xenopus as a developmental model and, as such, the transposon transgenesis method can be performed without additional equipment or specialized methodologies. The methods for injecting Xenopus embryos are well documented in the literature so here we provide a step-by-step guide to other aspects of transposon transgenesis, including screening mosaic founders for germline transmission of the transgene and general husbandry considerations related to management of populations of transgenic frogs.

Background: The Class II DNA transposons are mobile genetic elements that move DNA sequence from one position in the genome to another. We have previously demonstrated that the naturally occurring Tol2 element from Oryzias latipes efficiently integrates its corresponding non-autonomous transposable element into the genome of the diploid frog, Xenopus tropicalis. Tol2 transposons are stable in the frog genome and are transmitted to the offspring at the expected Mendelian frequency., Results: To test whether Tol2 transposons integrated in the Xenopus tropicalis genome are substrates for remobilization, we injected in vitro transcribed Tol2 mRNA into one-cell embryos harbouring a single copy of a Tol2 transposon. Integration site analysis of injected embryos from two founder lines showed at least one somatic remobilization event per embryo. We also demonstrate that the remobilized transposons are transmitted through the germline and re-integration can result in the generation of novel GFP expression patterns in the developing tadpole. Although the parental line contained a single Tol2 transposon, the resulting remobilized tadpoles frequently inherit multiple copies of the transposon. This is likely to be due to the Tol2 transposase acting in discrete blastomeres of the developing injected embryo during the cell cycle after DNA synthesis but prior to mitosis., Conclusions: In this study, we demonstrate that single copy Tol2 transposons integrated into the Xenopus tropicalis genome are effective substrates for excision and random re-integration and that the remobilized transposons are transmitted through the germline. This is an important step in the development of 'transposon hopping' strategies for insertional mutagenesis, gene trap and enhancer trap screens in this highly tractable developmental model organism.

Transposon-based integration systems have been widely used for genetic manipulation of invertebrate and plant model systems. In the past decade, these powerful tools have begun to be used in vertebrates for transgenesis, insertional mutagenesis, and gene therapy applications. Sleeping Beauty (SB) is a member of Tc1/mariner class of transposases and is derived from an inactive form of the gene isolated from Atlantic salmon. SB has been used extensively in human cell lines and in whole animal vertebrate model systems such as the mouse, rat, and zebrafish. In this study, we describe the use of SB in the diploid frog Xenopus tropicalis to generate stable transgenic lines. SB transposon transgenes integrate into the X. tropicalis genome by a noncanonical process and are passed through the germline. We compare the activity of SB in this model organism with that of Tol2, a hAT (hobo, Ac1, TAM)-like transposon system., ((c) 2009 Wiley-Liss, Inc.)

The amphibian, Xenopus laevis has been an excellent developmental model for over half a century. The large egg size, external fertilization and simple husbandry make this frog an ideal tool to study early vertebrate development. The tetraploid genome and long generation time, however, has hindered the use of X. laevis in large-scale genetic efforts. A close West African relative, Xenopus tropicalis (also commonly known as Silurana tropicalis), overcomes the limitations of X. laevis in genetic studies as X. tropicalis has a diploid genome and breeding adults can be obtained in six to nine months. We have focused our efforts on developing transposon systems for efficient transgenesis and insertional mutagenesis in the frog. Transposon systems have been used for transgenesis in a wide variety of model organisms. In this review, we will discuss the advantages and limitations of different transposon systems for generating transgenic Xenopus. In addition, we will describe strategies for the identification of novel genes through an insertional mutagenesis approach using transposable elements.

The generation of transgenic lines is vital to many genetic strategies and provides useful reagents for cell labeling and lineage-tracing experiments. Transposon-based systems offer simple, yet robust, platforms for transgenesis in the frog. Here, we provide a protocol for a microinjection-based transposon transgenesis method using a 'natural breeding' strategy for the collection of Xenopus tropicalis embryos. This method uses co-injection of a plasmid containing a transposon substrate together with synthetic mRNA encoding the transposase to achieve efficient integration of the transgene in the frog genome. We also describe a modified extension primer tag selection linker-mediated PCR technique to identify transposon integration sites within the host genome. This cloning strategy allows rapid identification of genomic sequences flanking the integration sites and multiple independently segregating transposon integration events in a single tadpole can be cloned simultaneously.

The study of amphibian embryogenesis has provided important insight into the mechanisms of vertebrate development. The frog Xenopus laevis has been an important model of vertebrate cell biology and development for many decades. Genetic studies in this organism are not practical because of the tetraploid nature of the genome and the long generation time of this species. Recently, a closely related frog, namely Xenopus tropicalis, has been proposed as an alternative system; it shares all of the physical characteristics that make X. laevis a useful model but has the advantage of a diploid genome and short generation time. The rapid accumulation of genetic resources for this animal and the success of pilot mutagenesis screens have helped propel this model system forward. Transposable elements will provide invaluable tools for manipulating the frog genome. These integration systems are ideally suited to transgenesis and insertional mutagenesis strategies in the frog. The high fecundity of the frog combined with the ability to remobilize transposon transgenes integrated into frog genome will allow large-scale insertional mutagenesis screens to be performed in laboratories with modest husbandry capacities.

The diploid frog Xenopus tropicalis is becoming a powerful developmental genetic model system. Sequencing of the X. tropicalis genome is nearing completion and several labs are embarking on mutagenesis screens. We are interested in developing insertional mutagenesis strategies in X. tropicalis. Transposon-mediated insertional mutagenesis, once used exclusively in plants and invertebrate systems, is now more widely applicable to vertebrates. The first step in developing transposons as tools for mutagenesis is to demonstrate that these mobile elements function efficiently in the target organism. Here, we show that the Medaka fish transposon, Tol2, is able to stably integrate into the X. tropicalis genome and will serve as a powerful tool for insertional mutagenesis strategies in the frog.

Cytokine signaling plays an important role in the survival and differentiation of vertebrate hematopoietic cells. In red blood cells, erythropoietin is a key component of the differentiation program and maintains the homeostasis of the erythroid compartment. In the adult, anemia stimulates high levels of circulating erythropoietin that drives erythropoiesis to restore normal levels of red blood cells in circulation. Erythropoietin activates the erythropoietin receptor on immature red blood cell precursors to promote their survival and differentiation. Although extensively studied in mammalian systems, a complete understanding of the function of the erythropoietin receptor during primitive erythropoiesis has been lacking. To address this problem, we have cloned the Xenopus laevis erythropoietin receptor in order to further understand the development of primitive erythropoiesis. The amphibian erythropoietin receptor shares 33% amino acid sequence identity with the mammalian erythropoietin receptors and contains the conserved extracellular ligand binding and fibronectin domains, the WSXWS motif common to cytokine receptors, and several tyrosine phosphorylation sites located on the intracellular domain of the receptor. Expression of the erythropoietin receptor is first detected by in situ hybridization in the ventral blood island during tailbud stages.

The Antarctic icefishes (family Channichthyidae, suborder Notothenioidei) constitute the only vertebrate taxon that fails to produce red blood cells. These fishes can be paired with closely related, but erythrocyte-producing, notothenioids to discover erythropoietic genes via representational difference analysis. Using a B30.2-domain-encoding DNA probe so derived from the hematopoietic kidney (pronephros) of a red-blooded Antarctic rockcod, Notothenia coriiceps, we discovered a related, novel gene, bloodthirsty (bty), that encoded a 547-residue protein that contains sequential RING finger, B Box, coiled-coil, and B30.2 domains. bty mRNA was expressed by the pronephric kidney of N. coriiceps at a steady-state level 10-fold greater than that found in the kidney of the icefish Chaenocephalus aceratus. To test the function of bty, we cloned the orthologous zebrafish gene from a kidney cDNA library. Whole-mount in situ hybridization of zebrafish embryos showed that bty mRNA was present throughout development and, after the mid-blastula transition, was expressed in the head and in or near the site of primitive erythropoiesis in the tail just prior to red cell production. One- to four-cell embryos injected with two distinct antisense morpholino oligonucleotides (MOs) targeted to the 5'-end of the bty mRNA failed to develop red cells, whereas embryos injected with 4- and 5-bp mismatch control MOs produced wild-type quantities of erythrocytes. The morphant phenotype was rescued by co-injection of synthetic bty mRNA containing an artificial 5'-untranslated region (UTR) with the antisense MO that bound the 5'-UTR of the wild-type bty transcript. Furthermore, the expression of genes that mark terminal erythroid differentiation was greatly reduced in the antisense-MO-treated embryos. We conclude that bty is likely to play a role in differentiation of the committed red cell progenitor.

As reported previously, AML1-ETO knock-in mice were generated to investigate the role of AML1-ETO in leukemogenesis and to mimic the progression of t(8;21) leukemia. These knock-in mice died in midgestation because of hemorrhaging in the central nervous system and a block of definitive hematopoiesis during embryogenesis. Therefore, they are not a good model system for the development of acute myeloid leukemia. Therefore, mice were generated in which the expression of AML1-ETO is under the control of a tetracycline-inducible system. Multiple lines of transgenic mice have been produced with the AML1-ETO complementary DNA controlled by a tetracycline-responsive element. In the absence of the antibiotic tetracycline, AML1-ETO is strongly expressed in the bone marrow of AML1-ETO and tet-controlled transcriptional activator double-positive transgenic mice. Furthermore, the addition of tetracycline reduces AML1-ETO expression in double-positive mice to nondetectable levels. Throughout the normal murine lifespan of 24 months, mice expressing AML1-ETO have not developed leukemia. In spite of this, abnormal maturation and proliferation of progenitor cells have been observed from these animals. These results demonstrate that AML1-ETO has a very restricted capacity to transform cells. Either the introduction of additional genetic changes or the expression of AML1-ETO at a particular stage of hematopoietic cell differentiation will be necessary to develop a model for studying the pathogenesis of t(8;21).

AML1 plays a critical role during hematopoiesis and chromosomal translocations involving AML1 are commonly associated with different forms of leukemia, including pre-B acute lymphoblastic leukemia. To understand the function of AML1 during B cell differentiation, we analyzed regulatory regions of B cell-specific genes for potential AML1-binding sites and have identified a putative AML1-binding site in the promoter of the B cell-specific tyrosine kinase gene, blk. Gel mobility shift assays and transient transfection assays demonstrate that AML1 binds specifically to this site in the blk promoter and this binding site is important for blk promoter activity. Furthermore, in vitro binding analysis revealed that the AML1 runt DNA-binding domain physically interacts with the paired DNA-binding domain of BSAP, a B cell-specific transcription factor. BSAP has been shown previously to be important for B cell-specific regulation of the blk gene. Physical interaction of AML1 with BSAP correlates with functional cooperativity in transfection studies where AML1 and BSAP synergistically activate blk promoter transcription by more than 50-fold. These results demonstrate physical and functional interactions between AML1 and BSAP and suggest that AML1 is an important factor for regulating a critical B cell-specific gene, blk.

Acute myeloid leukaemia (AML) is a major haematopoietic malignancy characterized by the proliferation of a malignant clone of myeloid progenitor cells. A reciprocal translocation, t(8;21)(q22;q22), observed in the leukaemic cells of approximately 40% of patients with the M2 subtype of AML disrupts both the AML1 (CBFA2) gene on chromosome 21 and the ETO (MTG8) gene on chromosome 8 (refs 3-5). A chimaeric protein is synthesized from one of the derivative chromosomes that contains the N terminus of the AML1 transcription factor, including its DNA-binding domain, fused to most of ETO, a protein of unknown function. We generated mice that mimic human t(8;21) with a "knock-in' strategy. Mice heterozygous for an AML1-ETO allele (AML1-ETO/+) die in midgestation from haemorrhaging in the central nervous system and exhibit a severe block in fetal liver haematopoiesis. This phenotype is very similar to that resulting from homozygous disruption of the AML1 (Cbfa2) or Cbfb genes, indicating that AML1-ETO blocks normal AML1 function. However, yolk sac cells from AML1-ETO/+ mice differentiated into macrophages in haematopoietic colony forming unit (CFU) assays, unlike Cbfa2-/- or Cbfb-/-cells, which form no colonies in vitro. This indicates that AML1-ETO may have other functions besides blocking wild-type AML1, a property that may be important in leukaemogenesis.

Further functional and biochemical characterization of the nuclear factor(s) which interacts with the EOS1 enhancer-like element in the IL-5R alpha promoter is currently in progress. Since different transcription factors recognize and interact with DNA in distinct fashions and with distinct structural motifs, we have modeled potential binding of the EOS1 factor to its cis-element based upon its methylation interference pattern (Fig. 2), using a cylindrical DNA helical projection (Fig. 6). Over a length of two helical turns, all nuclear protein contacts indicated by methylation interference map to one side of the DNA helix, suggesting that EOS1 binds in the major groove, across the minor groove, and on only one side of the helix. Further review of the model also reveals a potential diad symmetry for the binding site, suggestive of binding by a homodimer and consistent with the formation of the two DNA-protein complexes in our electrophoretic mobility shift experiments that could represent interactions with monomer versus dimer. Comparison of the EOS1 binding motif to similar models for the binding of other transcription factor families for which structural crystallographic and/or binding data is available suggests a similarity of the EOS1 complex to that of the bacterial helix-turn-helix phage lambda and 434 repressor-operator complexes, and the Cys4 zinc finger glucocorticoid response element (GRE) DNA-binding motifs, all of which show similar diad symmetry and binding in the major groove on one side of the DNA. The possibility that EOS1 functions as a GRE is being investigated, especially since there is a consensus AP-1 site at bp -440 to -432 of the IL-5R alpha promoter, immediately adjacent to the EOS1 binding site (see Fig. 5 in reference [36]) and AP-1/GRE interactions have been identified for composite response elements in the regulation of a number of different genes. The identification or cloning of EOS1, a potentially novel and eosinophil lineage-active transcription factor, should enhance our understanding of the processes involved in eosinophil development in particular and myeloid lineage commitment and differentiation in general.

The molecular basis for the commitment of multipotential myeloid progenitors to the eosinophil lineage, and the transcriptional mechanisms by which eosinophil-specific genes are subsequently expressed and regulated during eosinophil development are currently unknown. Interleukin-5 (IL-5) is a T cell and mast cell-derived cytokine with actions restricted to the eosinophil and closely related basophil lineages in humans. The high affinity receptor for IL-5 (IL-5R) is composed of an alpha subunit (IL-5R alpha) expressed by the eosinophil lineage, that associates with a beta c subunit shared with the receptors for IL-3 and granulocyte-macrophage colony stimulating factor (GM-CSF). As a prerequisite to studies of the transcriptional regulation of the IL-5R alpha subunit gene, we used three different methods, including primer extension, RNase protection, and 5'-RACE to precisely map the transcriptional start site to a position 15 base pairs (bp) upstream of the 5' end of the published sequence of IL-5R alpha exon 1. To initially identify the IL-5R alpha promoter, 3.5 kilobases (kb) and 561 bp of the 5' sequence flanking the transcriptional start site were subcloned into the promoterless pXP2-luciferase vector. Transient transfection of these constructs into an eosinophil-committed HL-60 subline, clone HL-60-C15, induced the expression of approximately 240-fold greater luciferase activity than the promoterless vector, identifying a strong functionally active promoter region within the 561 bp of sequence proximal to the transcriptional start site and with activity equivalent to pXP2 constructs containing the entire 3.5 kb of upstream sequence. To more precisely localize the cis-acting regulatory elements in this region important for promoter activity, a series of 5' deletion mutants of the 561-bp region were generated in the pXP2-luciferase vector. Deletion of the region between bp -432 and -398 reduced promoter activity by more than 80% in the HL-60-C15 cell line. Further analyses of the activity of the IL-5R alpha promoter constructs in various other eosinophil, myeloid, and non-myeloid cell lines indicated that the promoter was relatively myeloid and eosinophil lineage-specific in its expression. Consensus sequences for known transcription factor binding sites were not present in the 34-bp region of the promoter required for maximal activity, suggesting unique myeloid- and possibly eosinophil-specific regulatory elements.(ABSTRACT TRUNCATED AT 400 WORDS)